In mass spectrometry (MS), the ability to detect all analytes present in a sample depends on a number of parameters, including the complexity of the sample mixture. Ideally, the goal in any MS experiment is to detect 100% of the analytes present. However, as sample complexity increases, the ability to detect all species present markedly decreases. This is due to several factors, including: (1) ionization suppression (seen in MALDI (matrix-assisted laser desorption/ionization spectroscopy), (2) differences in ionization potential (seen in MALDI and ESI (electrospray ionization mass spectroscopy) and (3) the fact that higher abundance species can drown out the lower abundance species due to the limited dynamic range of common detectors (seen in MALDI and ESI). In MS, an analyte (e.g. peptide, protein, lipid, etc) must become ionized in the sample source region in order for it to reach the detector. The potential for any analyte to become ionized (ionization potential) is related to the sequence of the peptide (e.g. number of charged residues) as well as the presence of other components in the sample mixture, since other peptides may compete for ionization and contaminant adducts (e.g. Na, K) can adversely affect the ionization efficiency. These challenges are problematic in the field of proteomics, where any one sample may contain hundreds of proteins present in concentrations that span the dynamic range of 109 orders of magnitude (i.e. 108 log difference in abundance from the lowest abundance protein to the highest abundance protein). When these samples are subjected to enzymatic or chemical digestion, the resulting peptide mixtures are considerably more complicated than the original protein mixtures. Consequently, the presence of high abundance proteins in a proteomics mixture can present challenges for the detection of lesser abundant proteins due to resulting dynamic range issues and competition for ionization.
In addition to the adverse effects of high abundance peptides on the ionization efficiency and detection of other peptides, the presence of peptides from contaminating proteins in a proteomics study can affect the random match probability for peptide mass fingerprinting (PMF). In PMF, the peptide masses from an enzymatic or chemical digestion of the protein are compared to the masses from an in silico digest of protein in a database, for the purpose of protein identification. Consequently, when contaminant peptide masses (from keratin or trypsin, for example) are present, they may cause random matching of experimental masses to the theoretical masses in the database if they are combined with peptide analyte masses in a single search. Thus, the presence of peptides from both high abundance proteins and contaminant proteins can have an adverse-affect on (1) the ability to obtain complete sequence coverage of the protein(s) of interest and (2) can interfere with the ability to correctly identify the analyte of interest.
In proteomics, two approaches are commonly used to overcome complications from high abundance proteins or interference from contaminant proteins. These include (1) removal of peptide masses attributed to contaminant/high abundance proteins from the peptide peak list prior to database searching, or alternatively, filtering out peptides attributed to the contaminant/high abundance proteins after the database search and (2) removal of high abundance proteins as a whole, by affinity depletion (or other) methods prior to enzymatic/chemical digestion. Unfortunately, the removal of peptide masses from the peak lists, either prior to or after database searching, does not address the fundamental issues of ionization suppression or saturation of the detector that occur during data acquisition. While this approach may simplify the database search and data analysis, it does not lead to an ability to actually detect any more peptides. Additionally, the removal of intact proteins prior to digestion is plagued by the problem that protein depletion methods can non-specifically remove other proteins in low abundance (or high abundance proteins if there are high affinity interactions). Therefore, the removal of intact higher abundance proteins is disadvantageous for studies that aim to identify as many proteins as possible in the original sample.
In diagnostic assays for proteins of interest, the primary limitation is the detection capabilities of the target of interest. The most sensitive assays currently in use are generally those employing Enzyme Linked Immuno-Sorbant Assay (ELISA), which uses an antibody to capture a target and then a secondary antibody coupled to an enzyme to allow for amplification of the detection signal. These assays typically allow for up to low picogram levels of detection.